Preparation method of hair follicle tissue activity maintaining nutrient solution

Abstract

The invention belongs to the technical field of biomedical treatment, and particularly relates to a preparation method of a hair follicle tissue activity maintaining nutrient solution which comprises an umbilical cord mesenchymal stem cell conditioned culture medium. The invention relates to a hair growth liquid, which comprises insulin, hydrocortisone, vitamin B2, vitamin B6 and vitamin C. The prepared hair follicle tissue activity maintaining nutrient solution can effectively maintain the survival rate of hair follicles, the basic solution of the formula comes from all hUC-MSCs cell products, the safety problems such as cell carcinogenesis do not exist, the safety of the hair follicle tissue activity maintaining nutrient solution can be higher, and harm to the human body is avoided; moreover, the formula is externally applied to hUC-MSCs cell products, basically does not cause immune response, and is not limited to autologous stem cells, so that the formula has the advantages that the source is convenient, the standardized quality control and large-scale production can be realized in industrial production, and the like.

Description

Technical Field

[0001] The invention belongs to the field of biomedical technology, and in particular relates to a method for preparing a nutrient solution for maintaining hair follicle tissue activity. Background Art

[0002] The increasing pace of life and workplace competition have led to more and more young people suffering from hair loss. In addition to genetic and living environment factors, trauma, surgery, disease and other reasons can also cause hair loss in patients.

[0003] There is currently no effective treatment for hair loss, which can be divided into local, systemic, and other treatments. For local treatment, 2%-5% minoxidil tincture can be used, which can mainly reduce hair loss and stimulate new hair growth. It has a certain effect on mild to moderate patients with a short course of disease, small skin lesions, and sparse hair. For systemic medication, 1 mg of finasteride tablets can be taken orally every day to effectively slow down hair loss, increase hair volume, and accelerate hair growth. However, it takes a long time to take effect, requires long-term use of drugs, and has certain side effects. Surgical treatment refers to the use of donor hair follicles directly transplanted to the patient's bald area. This method has the advantages of high survival rate and no recurrence. Therefore, the number of patients requiring hair transplantation in clinical practice is increasing.

[0004] The in vitro preservation of hair follicles is of great significance for clinical hair transplantation, and the key lies in how to improve the survival rate of hair follicles and reduce the loss rate of hair follicles. Separation and preservation technology, transplantation technology, and postoperative management are three key factors in the hair transplantation process. Among them, how to improve the pre-transplantation activity of hair follicles is crucial to the entire process. Hair follicle transplantation surgery often requires a long time to operate, but in current clinical work, hair follicles are still stored in compound sodium chloride injection or low-temperature compound sodium chloride injection after being removed from the body, which can no longer meet the current clinical surgical needs. Therefore, the existing activity-maintaining nutrient solution is improved to provide a hair follicle tissue activity-maintaining nutrient solution. Summary of the invention

[0005] The purpose of the present invention is to provide a method for preparing a hair follicle tissue activity maintaining nutrient solution with a reasonable design and great application value for improving the survival rate of hair follicle transplantation, so as to solve the problems existing in the prior art.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] A nutrient solution for maintaining hair follicle tissue activity, comprising:

[0008] Umbilical cord mesenchymal stem cell conditioned medium;

[0009] A hair growth liquid comprising insulin, hydrocortisone, vitamin B2, vitamin B6 and vitamin C.

[0010] As a preferred embodiment, the present invention also provides a method for preparing a nutrient solution for maintaining hair follicle tissue activity, the steps of which include:

[0011] Step (i): To isolate hUC-MSCs, collect the umbilical cord of a newborn and cut it into 3-5 cm small segments. Then, wash the small segments of the umbilical cord with physiological saline, remove the epidermis and blood vessels, and retain the Wharton's jelly.

[0012] Step (2): Cut the Wharton glue obtained in step (1) into pieces of 0.5-1.0 mm 3 The tissue blocks were then evenly spread in a 15 cm culture dish;

[0013] Step (iii): Place the culture dish in step (ii) in a standard incubator at 37°C, saturated humidity, and 5% CO2 by volume. After 1.5 hours, add 10 mL of α-MEM medium containing 5% serum replacement and place in the incubator. Change the medium every 6 days. When the cell clones grow to a confluence of about 80%, use 0.05% trypsin to digest and passage.

[0014] Step (iv): Take the third generation hUC-MSCs in good condition, digest them with trypsin, count and dispense them into flow cytometry tubes, with about one million cells in each tube, wash them with PBS, resuspend them with antibody diluent, add the corresponding flow cytometry direct-labeled antibodies, incubate them at room temperature in the dark for 1 hour, and then detect them by flow cytometry;

[0015] Step (V): When the cells of P4 generation human umbilical cord mesenchymal stem cells reach 80%-90% fusion, the supernatant during the expansion period is collected, centrifuged to remove the cell components, and stored at -80°C for later use;

[0016] Step (six): based on the volume of 1000 mL of hair follicle tissue activity maintenance preservation solution, take 1000 ml of the culture supernatant, add the hair growth solution, and adjust the pH to 7.4 by adding sodium hydroxide or hydrochloric acid to obtain a composite preservation solution;

[0017] Step (seven): The composite preservation solution obtained in step (six) is filtered and sterilized using a bacterial filter to obtain a nutrient solution for maintaining hair follicle tissue activity.

[0018] As a preferred embodiment, the detection results in step (iv) show that CD105, CD90 and CD73 are all positively expressed in MSCs, and the proportion of positive cells is more than 90%, while the expressions of HLA-DR and CD45RA are both negative.

[0019] As a preferred embodiment, the hair growth solution in step (six) specifically comprises 30 IU / L insulin, 0.8 mg / L hydrocortisone, 10 mg / L vitamin B2, 50 mg / L vitamin B6 and 300 mg / L vitamin C.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] 1. The hair follicle tissue activity maintaining nutrient solution prepared by the technical solution of the present invention can effectively maintain the survival rate of hair follicles and has a significant effect on improving the activity of hair follicle tissue;

[0022] 2. Moreover, the basic liquid of this formula is derived from the products of hUC-MSCs cells, and there is no potential safety hazard such as cell carcinogenesis. It can make the hair follicle tissue activity-maintaining nutrient solution safer and avoid harm to human health;

[0023] 3. The hUC-MSCs cell product for external use in this formula basically does not cause immune response and rejection, and is not limited to autologous stem cells. Therefore, it has the advantages of convenient source, standardized quality control and large-scale production in industrial production. DETAILED DESCRIPTION

[0024] The embodiments described below are only some of the embodiments of the present invention and do not represent all the embodiments consistent with the present invention. The exemplary embodiments are now described as follows:

[0025] The hair follicle tissue activity maintaining nutrient solution of the present invention comprises:

[0026] Umbilical cord mesenchymal stem cell conditioned medium;

[0027] A hair growth liquid comprising insulin, hydrocortisone, vitamin B2, vitamin B6 and vitamin C.

[0028] As a preferred embodiment, the present invention also provides a method for preparing a nutrient solution for maintaining hair follicle tissue activity, the steps of which include:

[0029] Step (i): To isolate hUC-MSCs, collect the umbilical cord of a newborn and cut it into 3-5 cm small segments. Then, wash the small segments of the umbilical cord with physiological saline, remove the epidermis and blood vessels, and retain the Wharton's jelly.

[0030] Step (2): Cut the Wharton glue obtained in step (1) into pieces of 0.5-1.0 mm 3 The tissue blocks were then evenly spread in a 15 cm culture dish;

[0031] Step (iii): Place the culture dish in step (ii) in a standard incubator at 37°C, saturated humidity, and 5% CO2 by volume. After 1.5 hours, add 10 mL of α-MEM medium containing 5% serum replacement and place in the incubator. Change the medium every 6 days. When the cell clones grow to a confluence of about 80%, use 0.05% trypsin to digest and passage.

[0032] Step (iv): Take the third generation hUC-MSCs in good condition, digest them with trypsin, count and dispense them into flow cytometry tubes, with about one million cells in each tube, wash them with PBS, resuspend them with antibody diluent, add the corresponding flow cytometry direct-labeled antibodies, incubate them at room temperature in the dark for 1 hour, and then detect them by flow cytometry;

[0033] Step (V): When the cells of P4 generation human umbilical cord mesenchymal stem cells reach 80%-90% fusion, the supernatant during the expansion period is collected, centrifuged to remove the cell components, and stored at -80°C for later use;

[0034] Step (six): based on the volume of 1000 mL of hair follicle tissue activity maintenance preservation solution, take 1000 ml of the culture supernatant, add the hair growth solution, and adjust the pH to 7.4 by adding sodium hydroxide or hydrochloric acid to obtain a composite preservation solution;

[0035] Step (seven): The composite preservation solution obtained in step (six) is filtered and sterilized using a bacterial filter to obtain a nutrient solution for maintaining hair follicle tissue activity.

[0036] Based on the above, the detection results in step (iv) showed that CD105, CD90 and CD73 were all positively expressed in MSCs, and the proportion of positive cells exceeded 90%, while the expressions of HLA-DR and CD45RA were negative.

[0037] Based on the above, the hair growth solution in step (six) specifically contains 30 IU / L insulin, 0.8 mg / L hydrocortisone, 10 mg / L vitamin B2, 50 mg / L vitamin B6 and 300 mg / L vitamin C.

[0038] The working principle of the present invention is as follows:

[0039] Stem cells can secrete more than 300 kinds of biologically active protein molecules, and have strong functions in repairing skin damage and anti-aging of the skin. They are also rich in active substances such as growth factors, cytokines, interleukins, etc. necessary for hair follicle regeneration, so they can play an important role in hair follicle tissue. The hair follicle tissue activity maintenance nutrient solution prepared by the technical solution of the present invention can effectively maintain the survival rate of hair follicles. Moreover, the basic liquid of this formula comes from various hUC-MSCs cell products, and there is no safety problem such as cell carcinogenesis, which can make the hair follicle tissue activity maintenance nutrient solution safer and avoid harm to the human body; the external hUC-MSCs cell products of this formula basically do not cause immune response and rejection, and are not limited to stem cells of autologous origin, so they have the advantages of convenient source, standardized quality control and large-scale production in industrial production.

[0040] The above are only preferred specific embodiments of the present invention and are not intended to limit the scope of protection of the present invention. Any equivalent changes, modifications, substitutions and variations made by technicians in this technical field based on the concept of the present invention through logical analysis, reasoning or limited experiments on the basis of the prior art should be within the scope of protection determined by the claims.

Claims

1. A nutrient solution for maintaining hair follicle tissue activity, comprising: Umbilical cord mesenchymal stem cell conditioned medium; A hair growth liquid comprising insulin, hydrocortisone, vitamin B2, vitamin B6 and vitamin C.

2. The present invention also provides a method for preparing a nutrient solution for maintaining hair follicle tissue activity, the steps of which include: Step (i): To isolate hUC-MSCs, collect the umbilical cord of a newborn and cut it into 3-5 cm small segments. Then, wash the small segments of the umbilical cord with physiological saline, remove the epidermis and blood vessels, and retain the Wharton's jelly. Step (2): Cut the Wharton glue obtained in step (1) into pieces of 0.5-1.0 mm 3 The tissue blocks were then evenly spread in a 15 cm culture dish; Step (iii): Place the culture dish in step (ii) in a standard incubator at 37°C, saturated humidity, and 5% CO2 by volume. After 1.5 hours, add 10 mL of α-MEM medium containing 5% serum replacement and place in the incubator. Change the medium every 6 days. When the cell clones grow to a confluence of about 80%, use 0.05% trypsin to digest and passage. Step (iv): Take the third generation hUC-MSCs in good condition, digest them with trypsin, count and dispense them into flow cytometry tubes, with about one million cells in each tube, wash them with PBS, resuspend them with antibody diluent, add the corresponding flow cytometry direct-labeled antibodies, incubate them at room temperature in the dark for 1 hour, and then detect them by flow cytometry; Step (V): When the cells of P4 generation human umbilical cord mesenchymal stem cells reach 80%-90% fusion, the supernatant during the expansion period is collected, centrifuged to remove the cell components, and stored at -80°C for later use; Step (six): based on the volume of 1000 mL of hair follicle tissue activity maintenance preservation solution, take 1000 ml of the culture supernatant, add the hair growth solution, and adjust the pH to 7.4 by adding sodium hydroxide or hydrochloric acid to obtain a composite preservation solution; Step (seven): The composite preservation solution obtained in step (six) is filtered and sterilized using a bacterial filter to obtain a nutrient solution for maintaining hair follicle tissue activity.

3. The method for preparing a nutrient solution for maintaining hair follicle tissue activity according to claim 2, characterized in that: The detection results in step (iv) show that CD105, CD90 and CD73 are all positively expressed in MSCs, and the proportion of positive cells is more than 90%, while the expressions of HLA-DR and CD45RA are both negative.

4. The method for preparing a nutrient solution for maintaining hair follicle tissue activity according to claim 2, characterized in that: The hair growth solution in step (six) specifically comprises 30 IU / L insulin, 0.8 mg / L hydrocortisone, 10 mg / L vitamin B2, 50 mg / L vitamin B6 and 300 mg / L vitamin C.